Synthesis of 11-keto-delta**1,3,5(10)-estratrienes



- S P O r 3,488,365 Unltfid tatfis atnt Ce Patented Jan. 6, 1970 Grams SYNTHESIS ()F 1 xii gii ww-EsTRATRlENEs KZHPQ 1 Samuel c. Pan, Metuchen, Pacifico A. Princ p South Agar 20 River, Barbara Junta, Somerset, and Allen I. Cohen, East Brunswick, N.J., assignors to E. R. Squibb & Sons, Inc., New York, N.Y., a corporation of Delaware No Drawing. Filed Aug. 31, 1966, Ser. No. 576,254 Int. Cl. C07c 167/14, 169/10, 167/00 US. Cl. 260-39745 2 Claims ABSTRACT OF THE DISCLOSURE 11-keto-A -estratrienes are prepared from 10,6, 1IB-dihydroxy-l9-nor-A -androstenes by first oxidizing the dihydroxyandrostene to the corresponding lop-hydroxy-ll-keto derivative and then dehydrating such derivative.

This invention relates to and has for its object the provision of an improved process for preparing ll-keto-estranes l 1-keto-A -estratrienes) It has been found that a 10/8,11B-dihydroxy-l9-nor- A -androstene may be converted to an ll-ketoestrane derivative in high yield by a two-step process Without any substantial formation of undesired by-products. In essence, therefore, the process of this invention entails oxidizing a 105,1lfl-dihydroxy-19-nor-A -androstene, by treatment with an oxidizing agent such as chromic anhydride, to the corresponding 10 3-hydroxy-1 1-keto-19- nor-M-androstene, which are new compounds of this in vention; and dehydrating the latter, as by treatment with concentrated sulfuric acid in a glacial acetic acid medium. to yield the desired ll-ketoestrane final product.

Among the suitable starting steroids are included any of the 10,8,11B-dihydroxy-19-nor-A -androstenes. The preferred starting steroids, however, are the 3,17-dioxygenated 105,11fl-dihydroxy-19-nor-A -androstene, such as IOfiJIB-dihydroxy 19 nor A -androstene-3,17-dione, which is a new compound of this invention. To prepare these starting materials the corresponding IOB-hydroxyll-unsubstituted or 10,11-unsubstituted 19-nor-A -androstone is subjected to the action of enzymes of an 11B-hydroxylating microorganism, the reaction being carried out in the usual manner by culturing the microorganism in the presence of the steroid, or by treating the steroid with non-proliferating cells of the microorganism, or by intermixing the steroid with 11/3-hydroxylating enzymes previously obtained from the microorganism. The conditions for such microbial reaction are well known in the art and are similar to those specified in US. Patent No. 3,- 179,698.

The preferred llfi-hydroxylating microorganism that can be used as the source of the llfi-hydroxylating enzyme is Curvularia lunata.

The following examples illustrate the invention (all temperatures being in Centigrade):

EXAMPLE 1 105,1 lp-dihydroxy-19-nor-A -androstene-3 17 -dione (i) PREPARATION OF 1OB,11B-DIHYROXY-19-NOR-N- ANDROSTENE-3,17-DIONE A. Fermentatin.-Surface growth from each of three l0-day-old agar slant cultures of Curvularia: lunata (QM- l20-L), Army Quartermaster, Natick, Mass., the slant containing as a nutrient medium (A):

Distilled water to 1 liter.

is suspended in 5 ml. of a 0.01% sodium lauryl sulfate aqueous solution. One ml. portions of the suspension are used to inoculate ten 250 ml. conical flasks, each containing 50 ml. of the following sterilized medium (B):

Distilled Water to 1 liter.

After 72 hours of incubation at 25 with continuous rotary agitation (280 cycles per minute; 2 inch stroke), 10% vol./vol. transfers are made to forty 250 ml. conical flasks, each containing 50 ml. of fresh sterilized medium B plus 500 micrograms/ml. of 19-nor-A -andro stene-3,17-dione. The steroid is added by supplementing each flask with 0.25 ml. of a sterile solution of the steroid in N,N-dimethylformamide containing mg./ml. of steroid. A total of 1.0 gram is used. After 5 days of further incubation, the contents of the flasks are pooled through a Seitz clarifying pad. The flasks, mycelium and pads are washed with successive 50 ml. portions of warm water. The combined filtrate and washings have a volume of 2,000 ml.

B. Isolation and Characterization. -The combined filtrate and washings (2,000 ml.) are extracted three times with 500 ml. portions of methyl isobutyl ketone. The combined methyl isobutyl ketone extracts are washed with water, dried over anhydrous sodium sulfate and concentrated to dryness under vacuum, leaving about 300 mg. of crude product. This material is chromatographed ale 3592, 3450 (br.), 1733, 1669, 1600 cm.- NMR signals: 1-=8.81 p.p.m. (18CH -r=5.28 p.p.m. (11uH); 7:4.22 p.p.m. 441 -r=6.61 p.p.m. (H-bonded 1op OH and 11,s oH

(ii) ALTERNATIVE PREPARATION OF 10B,11B-DIHY- DROXY-lQ-NOR-N-ANDRO STENE-3, 17 -DIONE Following the procedure of step (i), but substituting an equivalent amount of IOB-hydroxy-19-nor-A -androstene-3,17-dione for the 19-nor-A -androstene-3,17-dione, 105,1 1fl-dihydroxy-19-nor-A -androstene-3, l7-dione having an M.P. of about 243 244 C. is obtained.

Moreover, by substituting the following steroid substrates for the 19-nor-A -androstene-3,17-dione in the procedure of Example 1, the indicated product is obtained:

4 A -androstene-3,11,17-trione, IOfi-hydroxy-ll-keto 19- nor-l7a-methyltestosterone and B-hydroxy-11-keto-19- Moreover, the following organisms can also be used to convert IOfl-hydroxy- 19 norandrostenedione to 10p 1 1 fl-dihydroxy-19-norandrostenedione:

Phycomyces blakesleeanus (CBS) (Centraalbureau voor schimmelcultures, Baarn, Netherlands) Coniothyrium helleb'ori (ATCC 12522) Corticium microsclerotia (NRLL 2727) EXAMPLE 2 IOfl-hydroxy-l l-keto-19-nor-A -androstene-3,17-dione To 10 ml. of a solution of mg. of 1013,11B-dihydroxy-19-nor-A -androstene-3,17-dione in acetone is added 0.1 ml. of a reagent prepared by dissolving 20 g. of chromic anhydride and 32 g. of concentrated sulfuric acid in 100 ml. of water. After allowing the reaction mixture to stand at room temperature for 10 minutes, the excess chromic anhydride is reduced by the addition of one drop of 95% ethanol. The reaction mixture is then diluted with 40 ml. of water and the steroids are extracted three times with 10 ml. portions of chloroform. After evaporating 01f the chloroform, the residue is chromatographed on Silica Gel GF plate in exactly the same way as described above in Example 1. The main uvabsorbing band is eluted, partitioned and evaporated to dryness in the same manner to give pure IOB-hydroxy- 19-nor-A -androstene-3,1 1,17-trione.

,gggg 3506, 1753, 1703, 1692, 1635. NMR signals:

1'=9.07 p.p.m. (IS-CH -r=4.l8 p.p.m. (4-H); -r=6.32 p.p.m. (H-bond in IOB-OH and ll-keto); -r=7.49 p.p.m. (120: and 126-H) A313,, 232 m nor-17u-ethynyltestosterone, respectively.

EXAMPLE 3 ll-keto-estrone from 10,8-hydroxy-19-nor-A -androstene- 3,11,17-trione A solution of 20 mg. of 10B-hydroxy-19-nor-A -androstene-3,11,17-trione in 5 ml. of glacial acetic acid, containing 15 mg. of concentrated sulfuric acid is heated in a boiling water bath for 30 minutes. After cooling, the reaction mixture is diluted with 40 m1. of water and the steroids are extracted three times with 10 ml. portions of chloroform. The chloroform phase was dried over anhydrous sodium sulfate and evaporated down to dryness to give crystalline ll-keto-estrone. It is recrystallized from acetone-n-hexane to give 16 mg. of the pure product, M.P. 222224[oc] +384 (C. 0.21, ethanol).

This invention may be variously otherwise embodied within the scope of the appended claims.

What is claimed is:

1. A process for preparing ll-keto-estrone which comprises dehydrating 10 3-hydroxy-19-nor-A -androstene-3, 11,17-trione with a strong acid.

2. The process of claim 1, wherein the strong acid is concentrated sulfuric acid in glacial acetic acid.

References Cited UNITED STATES PATENTS 2,729,654 1/1956 COltOn 260397.4 3,294,646 12/1966 Smith et al. -51

OTHER REFERENCES Djerassi, Steroid Reactions, Holden-Day, Inc., 1963, p. 385.

LEWIS GOTTS, Primary Examiner ETHEL G. LOVE, Assistant Examiner US. Cl. X.R. 19551 

